The present invention relates to an antibody against the cleavage product of vimentin; a method for detecting apoptosis using this antibody; and a use for this antibody.
Apoptosis is cell death in multicellular organisms. Surplus cells generated during developmental processes, cells no longer needed in an adult, cells damaged by radiation or chemical substances, or dangerous cells such as tumor cells are led to cell death by apoptosis, thus removed from a body.
Caspases are proteolytic enzymes (protease) that play a key role during apoptosis. Research on apoptosis has rapidly expanded since the 1990s. One of the key factors to have promoted the research is the identification of a caspase family of proteases that involves execution of apoptosis (Thomberry, N. and Lazebnik, Y. (1998) Science, 281, 1312-1316). At least 10 or more members of the caspase family are identified in mammals. Caspases are also shown to be present as an inactive precursor in normal cells. When apoptosis is initiated for a cell to die, an initiator caspase in the caspase family activates itself by limited proteolysis (processing). The activated initiator caspase activates another caspase by partially cleaving it, the cleaved caspase activates another caspase, and the process continues one after another. This amplification cascade mechanism is thought to achieve the whole activation. All caspases cleave the C-terminal side of a specific aspartic acid residue in protein, but the cleavage efficiency of each member of the caspase family varies depending on amino acid sequences near the cleavage site.
Apoptosis triggered by the stimulation of the anti-Fas antibody is the best analyzed apoptosis and thought to play a central role among adults (Nagata, S. (1997) Cell 88, 355-365). Caspase-8, involving execution of apoptosis is first activated among the caspase family in cells after stimulation with the anti-Fas antibody, and functions as an initiator (Boldin, M. P., Goncharov, T. M., Goltsev, Y. V. and Wallach, D. (1996) Cell 85, 803-815; Muzio, M., Chinnaiyan, A. M., Kishkel, F. C., O""Rourke, K., Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J. D., Zhang, M., Gentz, R., Mann, M., Krammer, P. H., Peter, M. E. and Dixit, V. M. (1996) Cell, 85, 817-827).
Several methods for detecting activation of a caspase have been employed, such as 1) detecting processing of a caspase or activation using an antibody recognizing a caspase; or 2) measuring protease activity using a substrate analog. Any of these methods, however, have a drawback in that the ability to distinguish between members of caspase family is limited. In the method of 1), production of a specific antibody capable of recognizing both an inactive precursor and an active type is often difficult.
It is the objective of the present invention to provide an antibody against the cleavage product of vimentin that is a main component of an intracellular skeletal protein; a method for detecting apoptosis using said antibody; and the use of said antibody.
As a result of intensive and extensive research toward the above-mentioned objective, the inventors have finally found that vimentin in an apoptotic cell is specifically cleaved by caspase-8, and they have succeeded in producing an antibody capable of detecting the specific cleavage of vimentin.
That is, the present invention relates to an antibody that reacts with the cleavage product of vimentin but does not react with intact vimentin. The cleavage product of said vimentin is the one cleaved by action of caspase (e.g., caspase-8). The abovementioned antibody can be either a polyclonal antibody or a monoclonal antibody.
Further, the present invention relates to a method for detecting caspase activity or a method for detecting apoptosis, comprising allowing the above antibody to react with cleavage product of vimentin and detecting the resulting reaction product.
Furthermore, the present invention relates to a reagent for detecting apoptosis containing the above antibody.
The present invention will now be described in detail. This specification includes part or all of the contents as disclosed in the specification and/or drawings of Japanese Patent Application No. 11-193235, which is a priority document of the present invention.